| Auxis thazard,mainly distributing in the East China Sea and South China Sea,is one of the main species of skipjack in China.It has a large catch,low price and high nutritional value.However,due to its acidic meat quality,high proportion of dark meat,poor taste and other reasons,it has not been well developed and utilized.Based on the characteristics of material and the present situation of resource utilization of Auxis thazard,a kind of peptide chelated calcium with high yield,high calcium content and high absorptivity was prepared by Auxis thazard protein peptide and calcium chloride in view of the low bioavailability of calcium in the population.In this article,the Auxis thazard protein peptide powder was used as the source of chelated protein.Firstly,the composition characteristics of the material were analyzed,the effect of chelating conditions on the yield of calcium chelated by protein peptide and calcium chloride was studied.Then,the calcium-binding active peptide was separated and purified by gel chromatography and reversed high performance liquid chromatography,and the binding mode of peptide with calcium ion was analyzed.Finally,the animal model of low-calcium was established,and the effect of Auxis thazard protein peptide chelated calcium on bone growth in rats was investigated.The main contents and results were as follows:1.The contents of protein,fat,water and mineral in protein-peptide powder were determined.And the composition of mineral elements,amino acids and molecular weight distribution of Auxis thazard protein peptide were systematically analyzed.On this basis,the potential advantages of Auxis thazard protein peptide as a source of high quality peptide for preparing calcium peptide chelated calcium were discussed.The results showed that the protein content,fat content,moisture content and mineral element content of Auxis thazard protein peptide powder were 88.91%,0.13%,5.16%and 2.15%respectively.The content of total amino acid and essential amino acid were 94.81 g/100g and 37.64 g/100g respectively,the essential amino acid score was more than 1,the chemical score was 0.67and the amino acid index was 0.87.The molecular weight of protein peptides from 180 U to 5000 U and from 180 U to 1000 U accounted for 78.16%and 67.99%,respectively.Therefore,Auxis thazard protein peptide,which contained high protein,low fat and reasonable amino acid composition,was a high-quality nutritional base material.2.The effects of p H,chelating temperature,chelating time and the mass ratio of peptide to calcium on Auxis thazard protein peptide chelated calcium were studied.And the changes of protein peptide before and after chelated with calcium were comparing by using ultraviolet spectrum,thermogravimetric,X-ray diffraction spectra and scanning electron microscope-energy spectrometer.Result showed that the optimum conditions for preparing the peptide-calcium chelate were mass ratio of peptide to calcium 2:1,p H 9.0,temperature 50?and reaction time 30 min.Under these conditions,the yield of chelate reached 41.06%and the calcium mass fraction was 14.98%.The results of various characterizations confirmed that a new type of peptide chelated calcium was prepared though the reaction of Auxis thazard protein peptide and calcium chloride.3.Sephadex G-15 gel chromatography and RP-HPLC were used to isolate and purify a novel calcium-binding active peptide from Auxis thazard protein peptide.The amino acid sequence was identified as Glu-Pro-Ala-His?MW=453.3 Da?by Xevo G2-XS Q-TOF,and its calcium binding capacity was 76.8±4.5 mg/g.The binding mode of Glu-Pro-Ala-His with calcium ion was that the binding sites of calcium-binding peptide involved the carboxylic group of Glu,as well as the carboxylic group and the amino group of His.Based on this result,the hypothesized molecular model of peptide-calcium chelate was constructed.4.The effects of different concentrations of Auxis thazard peptide chelated calcium?50 mg/100g·d,100 mg/100·d,200 mg/100g·d?on bone growth in rats were studied by establishing a rat model of low-calcium.The observational indexes included growth and development,blood calcium and phosphorus content,apparent calcium absorption rate,bone volume fraction,bone mineral density and trabecular microstructure.The results showed that the rats'weight growth rate was 84.77%,the ALP activity was 79.56 U/L,the apparent calcium absorption rate was 78.64%.In addition,the bone volume fraction was36.29%,the bone mineral density of cortical bone and cancellous bone were 6303.39mg/cc and 4126.09 mg/cc respectively,and the number of trabeculae was 4.87 mm-1.This indicated that Auxis thazard peptide chelated calcium can promote the weight growth of rats,effectively inhibit the activity of ALP,and improve the absorption rate of calcium in rats.In addition,It also can increase the bone volume fraction and the bone density,improve the bone trabecular microstructure,and promote the growth and development of bone. |
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